Project Summary Triple-negative breast cancer (TNBC) accounts for ~10% of all the breast cancer cases, but its survival rate is lower due to the lack of effective targeted treatments. This underscores the importance of finding new treatments for therapy-resistant TNBC, which is further complicated by the disease heterogeneity. Unfortunately, success of targeted therapies in TNBC has been limited, partly due to the lack of a detailed and mechanistic understanding of the drivers of each disease subgroup. An important contributor to the TNBC pathobiology is Notch signaling. Hyperactive Notch signaling promotes tumor growth, increases chemotherapy resistance, decreases survival, and increases the chance of metastases. Although the biomarkers of the Notch-active TNBC subgroup and drugs to target Notch signaling have been recently developed, treating patients with Notch inhibitory agents has been ineffective to date, partly due to the limited understanding of how the Notch signaling controls these fundamental processes. An important direct consequence of Notch signaling is to activate key TNBC genes, including MYC, CCND1 and SOX9. MYC is one of the most important proto-oncogenes promoting tumor growth and survival. CCND1 controls cell division among other cellular processes. SOX9 increases metastatic potential. Despite their importance, existing drugs fail to directly target these proteins. We propose to leverage the regulatory relationships between Notch and its target genes to selectively and efficiently target them. In order to achieve this goal, we first need to understand the mechanisms by which Notch regulates MYC, CCND1, and SOX9 in TNBC. We propose to use cutting-edge functional genomics and chromatin conformation assays to elucidate their Notch-directed regulatory mechanisms at population and single-cell resolutions. To develop more potent therapeutic options, we plan to use the latest single-cell resolution technologies to discover how drug-resistant cells circumvent the effect of Notch inhibitory drugs and maintain the expression of these critical Notch targets. We plan to use this knowledge in the future to tailor therapeutic strategies for individual TNBC patients with activated Notch signaling, and in the process, hope to improve the survival of patients with this aggressive and difficult to treat form of breast cancer. !